Recently, we have shown that (S)-
N'-Nitrosonornicotine [(S)-NNN], the major form of NNN in tobacco products, is a potent oral cavity and esophageal
carcinogen in rats. To determine the early molecular alterations induced by (S)-NNN in the oral and esophageal mucosa, we administered the
carcinogen to rats in the
drinking water for 10 wk and global gene expression alterations were analyzed by
RNA sequencing. At a false discovery rate P-value < 0.05 and fold-change ≥2, we found alterations in the level of 39 genes in the oral cavity and 69 genes in the esophagus. Validation of
RNA sequencing results by qRT-PCR assays revealed a high cross-platform concordance. The most significant impact of exposure to (S)-NNN was alteration of genes involved in immune regulation (Aire, Ctla4, and CD80),
inflammation (Ephx2 and Inpp5d) and
cancer (Cdkn2a, Dhh, Fetub B, Inpp5d, Ly6E, Nr1d1, and Wnt6). Consistent with the findings in rat tissues, most of the genes were deregulated, albeit to different degrees, in immortalized oral keratinocytes treated with (S)-NNN and in non-treated premalignant oral cells and malignant oral and head and neck squamous cells. Furthermore, interrogation of TCGA data sets showed that genes deregulated by (S)-NNN in rat tissues (Fetub, Ly6e, Nr1d1, Cacna1c, Cd80, and Dgkg) are also altered in esophageal and head and neck
tumors. Overall, our findings provide novel insights into early molecular changes induced by (S)-NNN and, therefore, could contribute to the development of
biomarkers for the early detection and prevention of (S)-NNN-associated oral and
esophageal cancers. © 2016 Wiley Periodicals, Inc.